Molecularly cloned SIVmac155/T3 utilizes CXCR4 as a viral coreceptor and rapidly induces severe CD4+ T cell depletion in rhesus macaques. A 184-bp deletion of nef gene from SIVmac155/T3 results in a loss of virus infectivity in U87MG cells in vitro and an attenuation of viral pathogenicity in vivo. However, a rhesus macaque infected with the nef deletion mutant (SIVmac155/T3 delta nef) eventually developed severe CD4+ T cell depletion. The disease progression was associated with an emergence and selection of mutant virus, designated SIVmac543. SIVmac543, like SIVmac155/T3 having a full-length nef gene, productively replicated in U87MG cells expressing CD4 and CXCR. Our data indicated that mutations in the 3' region of SIVmac543 genome compensate for a function of full-length nef gene for virus replication in U87MG cells. The most remarkable mutation in the 3' region of SIVmac543 genome was an extensive deletion in the nef gene: In addition to the 184bp deletion introduced in SIVmac155/T3 delta nef, the SIVmac543 has a 299-bp deletion in the nef overlapping with U3 of the 3' LTR. The extensive additional deletion, however, restores the N- and C-terminal Nef polypeptides into a single translation frame, and the SIVmac543 nef is predicted to encode approximately 11 kDa myristylated protein. We hypothesize that the heavily deleted SIVmac543 nef gene encodes a novel Nef protein that contains a minimum domain(s) required for both virus replication in vitro and pathogenicity in vivo. The evolution and selection of the SIVmac543 nef gene might be responsible for the pathogenic conversion of attenuated SIV delta nef in vivo. To verify our hypothesis, three Specific Aims are proposed: In Aim 1, we will determine if the mutations in the SIVmac543 nef gene contribute virus replication in U87MG cells by construction of recombinants and site-specific mutants. The effect of the SIVmac543 nef gene on virus replication in rhesus CD4+ T cells, virion infectivity and viral expression will also be analyzed. In Aim 2, the biological functions of SIVmac543 Nef protein will be defined by examining its interactions with CD4, MHC-class 1, T-cell receptor, PAK kinases and Src-family tyrosine kinases. The significance of the heavily deleted nef gene of SIVmac543 for virus pathogenicity in vivo will be investigated using rhesus macaque infection studies (Aim 3). The proposed studies will shed a new light on the structure- function of Nef protein and potentially define the minimum domain of Nef protein required for virus pathogenicity.